Rationale for the real‐time and dynamic cell death assays using propidium iodide

Abstract

We have recently reported an innovative approach to use charged fluorochromes such as propidium iodide (PI) in the real-time, dynamic cell viability assays. The present study was designed to provide a mechanistic rationale for the kinetic assays using cell permeability markers. Uptake of PI by live cells, effect on the cell cycle, long term proliferation capacity, DNA damage response and pharmacologic interactions with anticancer drugs were studied using both laser scanning microscopy and laser scanning cytometry. Exposure of human carcinomic alveolar basal epithelial A549 cells in cultures to 1.5 or 7.5 µM of PI for 24 h had minimal effect on cell cycle progression including DNA replication as measured by incorporation of 5’-ethynyl-2-deoxyuridine (EdU) detected by the “click chemistry” approach and measured by laser scanning cytometry. A modest reduction, from 44% to 40% or 33%, in frequency of DNA replicating cells was seen after 48 h at 1.5 or 7.5 µM concentration of PI. There was no evidence of increased phosphorylation of histone γH2AX in cells growing in the presence of 1.5 or 7.5 µM of PI for up to 48 h. Confocal image analysis, of HeLa and NIH 3T3 mouse embryonic fibroblasts growing in the presence of PI showed granular distribution in cell cytoplasm suggesting PI accumulation in endosomes and progressive increase in fluorescence of nucleoli reflecting PI binding to nucleolar RNA. The overall responses of cells to cytotoxic agents were also not affected by the growth in the presence PI. Our data lend further support to the notion that propidium iodide can be effectively used in real-time, kinetic viability assays.

Real‐time flow cytometry for the kinetic analysis of oncosis

“…More recently the use of SYTO16 and viability dyes has allowed the discrimination of apoptosis and oncosis in that no reduction in SYTO16 was observed in cells undergoing oncosis compared to that observed in cells undergoing apoptosis (7). In another advance, propidium iodide has been used in a real-time image analysis study of apoptosis from the start of the induction process and continuously over a 24 h period (27)(28)(29). In this approach adherent cells were preloaded with Hoechst and grown in culture with a low concentration of PI (0.25 lg/ml) and then exposed to STS for 24 h with images taken every 15 min in a real-time manner (27).…”

“…Thus the measurement of mitochondrial membrane and plasma membrane potentials in real-time affords a rapid easy method to distinguish oncosis and apoptosis at the induction stage of these two different necrobiological processes. This approach to study oncosis could used in conjunction with that employed to study apoptosis in real-time and SYTO16 and thus enable differentiation between the induction of oncosis and apoptosis in vitro and potentially ex vivo (27)(28)(29).…”

“…More recently a major advance in the real-time study of apoptosis developed by Wlodkowic and colleagues has shown that a range of viability dyes, including PI and SYTO16 are not toxic to cells and can be used in Lab-on-a-Chip-based real-time imaging systems in the study of the induction of apoptosis by real-time fluorescence and morphological analysis (2,(27)(28)(29). In a similar vein a polarity-sensitive annexinbased biosensor (pSIVA) with switchable fluorescence states allows the real-time detection of apoptotic cells by time-lapse imaging from 5-42 h (30).…”

Microfluidics: Emerging prospects for anti-cancer drug screening

“…In this context, functional cytomics is slowly becoming an omnipotent part of the post-genomic drug discovery pipeline [7,8] . Although, it is widely recognized that the validation of therapeutic targets revealed by proteomic and genetic screens requires 4D (3D space plus time) functional cellbased assays, their widespread applications are still underdeveloped [1,9,10] . High-content analysis (HCA) is one of the key platforms that recently improved drug screening routines by collecting content-rich data sets [3] .…”

“…High-content analysis (HCA) is one of the key platforms that recently improved drug screening routines by collecting content-rich data sets [3] . Surprisingly, however, the commonly used HCA approaches are still based on a static principle, yielding information on cell status at a single time point [1,9,10] . Capabilities of high-speed, multiparameter and real-time analysis of great numbers of isolated cells are as yet profoundly limited [1,9,10] .…”